A major focus in the area of human cancer pertains to the programs involved in cell cycle exit, differentiation, and maintenance of the differentiated state. Research over nearly two decades has suggested that the retinoblastoma tumor suppressor (pRB) and two related proteins (p107 and p130; collectively referred to as pocket proteins) play a fundamental role in regulating the cell cycle, and pRB has been shown to be a prototypical tumor suppressor mutated in a large portion of human tumors. In addition, pRB has been shown to play a pivotal role in differentiation of several tissues, including muscle and bone. Our work during the previous funding period has highlighted unique roles for each of the pocket proteins in responding to growth arrest cues and in both promoting and maintaining differentiation of muscle. In addition, we have succeeded for the first time in purifying pRB complexes from proliferating and differentiated muscle cells. In this grant, we propose the following aims to further dissect the mechanisms underlying pocket protein involvement in cell cycle exit and differentiation. (1) We will characterize pRB complexes in proliferating and differentiated cells and examine the impact of depleting associated proteins on gene expression and differentiation; (2) we will identify targets of the pRB complexes in cultured cells and in muscle tissue and examine the role of pRB complexes in modification of target gene chromatin; and (3) we will examine whether mechanisms analogous to those discovered in Aims 1 and 2 pertain to reversible growth arrest as well and determine if there are mechanisms that distinguish reversible and irreversible cell cycle exit. These studies will enhance our understanding of regulatory controls that are essential for both reversible and permanent withdrawal from the cell cycle and differentiation. [unreadable] [unreadable] [unreadable]